A method for reducing nicotine contamination of a flue-cured tobacco field by interplanting crops and related applications and products

Abstract

The application relates to a method for reducing nicotine pollution of a flue-cured tobacco field on a interplanted crop and related applications and products, and belongs to the technical field of flue-cured tobacco field soil improvement. In the application scheme, the flue-cured tobacco field is treated by using biochar in cooperation with fly ash and wood vinegar, the soil of the flue-cured tobacco field can be improved, and the soil microbial environment can be changed, the absorption and biological accumulation of the interplanted crop on nicotine can be effectively reduced, the method has important significance for reducing the nicotine pollution of the flue-cured tobacco field on the interplanted crop and guiding how to interplant the crop in the flue-cured tobacco field, and has important industrial application value.

Description

Technical Field

[0001] This application relates to a method for reducing nicotine pollution of intercropped crops in flue-cured tobacco fields, as well as related applications and products, belonging to the field of flue-cured tobacco field soil improvement technology. Background Technology

[0002] In recent years, tobacco alkaloids have attracted global attention due to their potential ecological risks in agricultural ecosystems caused by their toxicity. Nicotine is a secondary metabolite widely found in nightshade plants, including potatoes and tomatoes, especially in tobacco, where it accounts for approximately 5% of the dry weight of tobacco and about 95% of the total alkaloids. The long half-life of nicotine in soil, the widespread use of commercially available nicotine as a broad-spectrum cytotoxic phytotoxic agent, and the year-round cultivation of tobacco in agricultural systems have led to ecological pollution. Globally, tobacco cultivation covers more than 4 million hectares, and nicotine concentrations detected in tobacco-growing soils have reached as high as 13.13 mg / kg. More than 95% of the nicotine in tobacco is converted to nornicotine through demethylation, a highly toxic pollutant to soil, plants, and the environment. Due to the horizontal transfer characteristics of these tobacco alkaloids, residual nicotine and nornicotine in the soil are easily absorbed by the roots and transferred to the edible organs of crops, thus posing a food safety risk to human health. Given the threat posed by nicotine to ecosystems and consumers, the EU has banned the use of plant-based pesticides derived from nicotine since 2009 and set a maximum residue limit of 0.01 mg / kg (dry weight) in food.

[0003] Intercropping flue-cured tobacco with appropriate crops not only increases the multiple cropping index of the field but also makes full use of light and temperature resources before winter, effectively increasing farmers' income. Peas are a typical global crop with an annual yield exceeding 36.5 tons. Rich in nutrients, with a good taste and diverse cooking methods, they are favored by consumers. As a traditional spring crop, peas are planted in close succession to tobacco, serving as a follow-up crop. In recent years, the practice of intercropping peas after tobacco has spread to 300,000 mu (approximately 20,000 hectares) in Yunnan Province, generating an annual output value of 1.8 billion yuan. Yunnan Province's annual tobacco planting area reaches 6 million mu (approximately 400,000 hectares), indicating significant development potential for this practice. However, previous studies have shown that in locations previously planted with tobacco, nicotine or other tobacco alkaloid extracts can affect plant growth through allelopathic effects. Research also indicates that the accumulation of residual nicotine in the soil affects the quality of broad beans, posing a significant potential food risk to human health. Edible pea plants grown in tobacco fields are easily contaminated by toxic tobacco alkaloids in the soil.

[0004] However, research on remediation technologies for toxic tobacco alkaloids in soil crop ecosystems is still limited. Therefore, to ensure the health of soil ecosystems and the safety of food crops, especially successor crops, there is an urgent need for an environmentally friendly, efficient, and economical soil remediation technology to reduce the absorption and accumulation of tobacco residual alkaloids in successor soil crop ecosystems. Summary of the Invention

[0005] To address the aforementioned issues, this application provides a method and related applications and products for reducing nicotine pollution in intercropped crops from flue-cured tobacco fields. The proposed solution utilizes biochar in combination with fly ash and wood vinegar, which can influence the dissipation of tobacco alkaloids in the soil by soil microorganisms, thereby improving nicotine pollution in nicotine-rich soils and reducing nicotine accumulation in intercropped crops in flue-cured tobacco fields. This has significant implications for guiding the intercropping of crops in flue-cured tobacco fields.

[0006] According to one aspect of this application, a method for reducing nicotine pollution of intercropped crops in flue-cured tobacco fields is provided, the method comprising the following steps:

[0007] S1. Preparation of cow dung biochar: Cow dung raw material is placed in a muffle furnace, and nitrogen gas flows into the vacuum furnace at a rate of 0.1-0.3 L / min for 5-15 min. Then the dried raw material is heated to 450-550℃ and held for 3-5 h. Pyrolytic carbon is prepared by slow pyrolysis to obtain cow dung biochar. The prepared cow dung biochar sample is then crushed and passed through a 2 mm sieve for later use.

[0008] S2. Apply 50±10 kg / mu of superphosphate and 25±5 kg / mu of 15:15:15 compound fertilizer as base fertilizer. Then apply cow manure biochar and fly ash together with the base fertilizer. At the same time, apply 1-3 kg / mu of chlorpyrifos granules. Use a hoe to gently mix the fertilizer and pesticides with the soil. Before the first irrigation, spray with wood vinegar solution diluted 8-12 times.

[0009] Optionally, the amount of cow dung biochar applied is 50-300 kg / mu, the amount of fly ash applied is 20-100 kg / mu, and the amount of wood vinegar applied is 10-100 kg / mu.

[0010] According to another aspect of this application, a method for intercropping peas in a flue-cured tobacco field is provided, the method comprising the following steps:

[0011] S1. Preparation of cow dung biochar: Cow dung raw material is placed in a muffle furnace, and nitrogen gas flows into the vacuum furnace at a rate of 0.1-0.3 L / min for 5-15 min. Then the dried raw material is heated to 450-550℃ and held for 3-5 h. Pyrolytic carbon is prepared by slow pyrolysis to obtain cow dung biochar. The prepared cow dung biochar sample is then crushed and passed through a 2 mm sieve for later use.

[0012] S2. Pretreatment of flue-cured tobacco fields: When the flue-cured tobacco has 4-5 leaves left at the top, prepare to sow peas. On the prepared soil surface, dig a shallow furrow 3-5 cm deep. Apply 50±10 kg / mu of superphosphate and 25±5 kg / mu of 15:15:15 compound fertilizer as base fertilizer in the furrow. Then apply cow manure biochar and fly ash together with the base fertilizer. At the same time, apply an additional 1-3 kg / mu of chlorpyrifos granules. Use a hoe to gently mix the fertilizer and pesticides with the soil. Before the first irrigation, spray with wood vinegar solution diluted 8-12 times.

[0013] S3. Pea planting: Sow 4-6 kg of pea seeds per mu, with a plant spacing of 4-6 cm in the prepared planting furrows; or, sow 2-3 seeds per hole with a hole spacing of 10-15 cm, and plant in single rows.

[0014] S4. When the peas grow to 15-20cm tall, build a trellis to guide the vines. First, remove the buds on the tobacco plants and leave the tobacco stalks 1-1.5m high as supports. Then, use yarn to pull a horizontal line 0.8-1m above the soil surface on each tobacco stalk. Tie the horizontal line to the tobacco stalk and use yarn to wrap and hang each pea plant on the horizontal line.

[0015] S5. After the vines are trained, shallowly cultivate the soil surface and leave shallow furrows near the roots of the plants. Apply a seedling fertilizer once with 15±5 kg / mu of urea diluted in water. Before the peas flower, apply 20±5 kg / mu of 15:15:15 compound fertilizer and 15±5 kg / mu of urea once more. During the flowering and pod-setting period of the peas, spray the leaves with 0.5% potassium dihydrogen phosphate 1-2 times and apply 20±5 kg / mu of 15:15:15 compound fertilizer diluted in water.

[0016] Optionally, the amount of cow dung biochar applied is 50-300 kg / mu, the amount of fly ash applied is 20-100 kg / mu, and the amount of wood vinegar applied is 10-100 kg / mu.

[0017] According to another aspect of this application, a method for reducing nicotine pollution of intercropped crops in flue-cured tobacco fields is provided, the method comprising the step of treating the flue-cured tobacco fields by returning biochar, fly ash and wood vinegar to the fields, wherein the biochar is cow dung biochar or straw biochar.

[0018] Optionally, the straw biochar is corn biochar.

[0019] Optionally, the method includes the step of treating the flue-cured tobacco field by returning cow manure biochar, fly ash, and wood vinegar to the field.

[0020] Optionally, the amount of cow dung biochar applied is 50-300 kg / mu, the amount of fly ash applied is 20-100 kg / mu, and the amount of wood vinegar applied is 10-100 kg / mu.

[0021] Optionally, the intercropping crop is peas.

[0022] Optionally, the edible portion of the intercropped pea crop contains reduced levels of tobacco alkaloids.

[0023] According to another aspect of this application, the application of cow dung biochar, fly ash, and wood vinegar in reducing nicotine pollution in flue-cured tobacco fields is provided.

[0024] According to another aspect of this application, the application of cow dung biochar, fly ash and wood vinegar in the intercropping of peas in flue-cured tobacco fields is provided.

[0025] According to another aspect of this application, a flue-cured tobacco field remediation fertilizer comprising cow manure biochar, fly ash, and wood vinegar is provided.

[0026] The beneficial effects of this application include, but are not limited to:

[0027] 1. The cow dung biochar group was more effective than the corn stalk biochar group in reducing the absorption and bioaccumulation of nicotine in peas;

[0028] 2. Both cow dung biochar and corn stalk biochar can reduce the absorption and accumulation of tobacco alkaloids in pea plants, and the cow dung biochar treatment is more efficient in reducing the absorption and accumulation of tobacco alkaloids.

[0029] 3. The cow dung biochar group reduced the translocation of nicotine and nornicotine to pea seedlings more effectively than the corn stalk biochar group;

[0030] 4. The cow dung biochar group increased the concentrations of nicotine and nornicotine in the rhizosphere soil more effectively than the corn straw biochar group;

[0031] 5. Cow dung biochar group was more effective than corn straw biochar group in reducing the concentration of nicotine and nornicotine in in-situ soil pore water;

[0032] 6. Both cow dung biochar and corn stalk biochar can prolong the duration of nicotine in unsterilized soil, and both cow dung biochar and corn stalk biochar can promote the dissipation of nicotine through abiotic degradation, with cow dung biochar showing better results.

[0033] 7. Compared with corn stalk biochar treatment, cow dung biochar treatment has a stronger nicotine adsorption capacity.

[0034] 8. Treatment with cow manure biochar increased the relative abundance of Proteobacteria, Gemmatimonadota, and Bacteroidota, while decreasing the relative abundance of Acidobacteriota, thereby enhancing the degradation of nicotine and / or nornicotine. Attached Figure Description

[0035] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0036] Figure 1 This is a graph showing the effects of cow dung biochar and corn straw biochar on the growth of pea plants in nicotine-contaminated soil, as described in Example 1 of this application.

[0037] Figure 2 The figure shows the effects of the cow dung biochar group and the corn straw biochar group involved in Test Example 1 of this application on the concentrations of nicotine (a) and nornicotine (b) in pea roots and stems, the translation factor (TF) (c) of nicotine and nornicotine in pea roots and aboveground parts, and the dry weight (d) of pea roots and aboveground parts (different lowercase letters indicate significant differences between different treatments (Duncan multiple comparison test, n=3, p<0.05(*),0.01(**)));

[0038] Figure 3 The figure shows the effect of cow dung biochar and corn straw biochar on the total absorption of nicotine and nornicotine in pea stems (a) and roots (b) in nicotine-contaminated soil, as described in Test Example 1 of this application (different lowercase letters indicate significant differences between different treatments (Duncan multiple comparison test, n=3, p<0.05(*),0.01(**))).

[0039] Figure 4 The results of Test Examples 2 and 3 of this application are shown in the figure (a), aboveground enrichment factor (SCF) of nicotine and nornicotine in pea soil, and pore water concentration of nicotine and nornicotine in pea soil (b), and (c) (different lowercase letters indicate significant differences between different treatments (Duncan multiple comparison test, n=3, p<0.05(*), 0.01(**))).

[0040] Figure 5 The graph shows the biological (a) and non-biological (b) degradation results of nicotine in the soil in the groups with or without cow manure biochar and corn straw biochar involved in Test Example 5 of this application (different lowercase letters indicate significant differences between different treatments (Duncan multiple comparison test, n=3, p<0.05(*),0.01(**)));

[0041] Figure 6The graph shows the changes in soil bacterial community composition after the addition of cow dung biochar and corn straw biochar in Test Example 6 of this application (a: percentage of abundance at the phylum level; b: percentage of abundance at the genus level). Detailed Implementation

[0042] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present application are all purchased through commercial channels.

[0043] Biochar is a carbon-rich material with a porous structure, abundant functional groups, and a large surface area. Its significant advantages in agricultural soil remediation are mainly reflected in improving soil structure, enhancing available nutrients, promoting enzyme activity, and strengthening the rhizosphere microbial community for plant growth. In recent years, biochar has been proven effective in reducing the uptake of various soil organic pollutants (pesticides, ARGs, polycyclic aromatic hydrocarbons, etc.) by plants. Biochar can remediate soil organic pollutants by reducing their bioavailability and promoting their adsorption, abiotic degradation, and biodegradation in the soil.

[0044] In soil, the dissipation of pollutants is governed by the interactions between soil, biochar, and the pollutants themselves, with the physicochemical properties of biochar being a key factor influencing the removal efficiency of organic pollutants in soil. Among the factors affecting biochar properties, the type of raw material is one of the most important influencing the fate of pesticides in soil. For example, Li et al. (2018b) found that, under the same conditions, three types of biochar prepared from crop weeds, sawdust, and rice husks showed significant differences in specific surface area, pH value, and nutrient content, and their adsorption capacity for acetochlor in soil also differed. Among the three types of biochar, the adsorption constant Kf value of acetochlor prepared from rice husks was 87 times and 136 times that of biochar prepared from weeds and sawdust, respectively, with a specific surface area of ​​54.95 m². 2 / g and pore volume 0.04cm³ 3 / g maximum.

[0045] The potential mechanisms by which biochar with different physicochemical properties affects the dissipation of soil pollutants vary. For example, Ali et al. (2019) reported that the addition of pyrolytic biochar from four types of agricultural waste, including sewage sludge, soybean straw, rice straw, and peanut shells, reduced the bioavailability of organochlorine pesticides in soil by 47–60%, and that changes in soil microbial community structure drove the degradation of organochlorine pesticides in the soil. Biochar produced from different raw materials has varying remediation efficiencies for soil pollutants, and biochar dissipates pollutants in soil through multiple pathways, determined by its physicochemical properties. However, current research has focused on the remediation of exogenous toxic substances, neglecting endogenous natural toxic products produced by donor plants.

[0046] Furthermore, biodegradation is another key factor influencing nicotine dissipation in soil. Various bacterial strains, including Pseudomonas, Achromobacter, Azospirillum, Mycolicibacterium, and Terrimonas, have great potential in degrading and reducing nicotine in soil. Previous studies have reported that biochar can significantly influence microbial diversity and bacterial community changes in soils contaminated with neonicotinoid pesticides, which have similar nicotine structures, by providing nutrients and improving the soil microenvironment. Therefore, the inventors hypothesize that biochar may affect the dissipation of tobacco alkaloids in soil by soil microorganisms, thereby improving nicotine pollution in nicotine-contaminated soils and addressing the nicotine pollution problem in intercropping tobacco fields currently existing in the field.

[0047] Furthermore, the production of biochar from agricultural waste such as poultry manure and crop straw has gained increasing attention in recent years as an efficient and sustainable strategy for remediating contaminated soils. Compared to plant-derived biochar, biochar produced from animal manure contains more nutrients, has a higher pH value, and a larger specific surface area. It should therefore possess a higher adsorption and / or degradation capacity in reducing the absorption and accumulation of nicotine and nornicotine in the soil by plants and promoting their dissipation in the soil. Currently, there is no research on the impact of biochar on the dissipation of tobacco alkaloids in soil-crop systems.

[0048] Researchers have found that using cow dung biochar or corn stalk biochar alone can have a certain effect on repairing the soil in flue-cured tobacco fields and reducing the nicotine content in the edible parts of pea plants. However, the improvement effect of using cow dung biochar or corn stalk biochar alone is limited, and there is still room for further improvement.

[0049] Therefore, in this application, by adding a certain proportion of cow manure biochar and corn straw biochar to nicotine-contaminated soil, combined with fly ash and wood vinegar, the nicotine content in pea plants was determined to achieve the following two basic objectives: 1) to evaluate the effects of cow manure biochar and corn straw biochar treatment on the absorption and accumulation of nicotine and nornicotine in peas; 2) to evaluate the effects and mechanisms of cow manure biochar and corn straw biochar on the degradation of nicotine and nornicotine in agricultural soils. This application is the first to report the effects of biochar on the translocation of nicotine and nornicotine from soil to plants, and the published results will provide fundamental information for developing biochar remediation technologies to remediate agricultural soils contaminated with naturally occurring toxic substances and protect consumer health.

[0050] In this application, the cow dung biochar and corn stalk biochar were prepared by the researchers. The cow dung and corn stalk waste were sourced from farmland at the Jimo Experimental Base of the Tobacco Research Institute of the Chinese Academy of Agricultural Sciences and the nearby Qingdao Aote Dairy Breeding Farm, respectively. Wood vinegar, a high-value acidic byproduct of biomass pyrolysis char production, was purchased from Henan Boton Biotechnology Co., Ltd. Fly ash possesses excellent physicochemical properties and can be widely used to improve heavy clay, raw soil, acidic soil, and saline-alkali soil, compensating for their deficiencies of being acidic, lean, and sticky. Fly ash contains a large amount of water-soluble nutrients essential for crops, such as silicon, calcium, magnesium, and phosphorus, and can therefore be used as agricultural fertilizer. It was purchased from Xiamen Yicai Fly Ash Co., Ltd. Pea seeds were purchased online (Shouhe Seed Industry). The 15:15:15 compound fertilizer is a commonly used balanced compound fertilizer in this field, with nitrogen, phosphorus, and potassium content all at 15%.

[0051] Example 1

[0052] CDBC (cow manure biochar) and MSBC (corn straw biochar) at addition rates of 1.5% and 3.0% (w / w) were thoroughly mixed with air-dried soil. Then, 1.50 kg soil samples with and without CDBC and MSBC were placed in plastic containers (15 × 12 cm, diameter × height). Next, 50.0 ml of 300 mg / L nicotine solution was added to the soil and mixed thoroughly, resulting in an initial concentration of 10.0 mg / kg. Five treatment groups were designed, each containing 2.0% (w / w) fly ash and 1.0% (w / w) wood vinegar: 1) CK: Nicotine-treated soil without any biochar, fly ash, or wood vinegar; 2) 1.5% CDBC: Nicotine-treated soil with 1.5% CDBC, 2.0% fly ash, and 1.0% wood vinegar; 3) 3% CDBC: Nicotine-treated soil with 3% CDBC, 2.0% fly ash, and 1.0% wood vinegar; 4) 1.5% MSBC: Nicotine-treated soil with 1.5% MSBC, 2.0% fly ash, and 1.0% wood vinegar; 5) 3% MSBC: Nicotine-treated soil with 3% MSBC, 2.0% fly ash, and 1.0% wood vinegar. Treatments 2) and 3) were cow dung biochar groups, and treatments 4) and 5) were corn straw biochar groups.

[0053] Each treatment was set up in triplicate. Before sowing, tap water was added to all pots every three days to bring the water-holding capacity (WHC) to 60% by weight, and the internal relative humidity (IRH) of the incubator was maintained at 60% (24.0 ± 2.0℃, 7 days). Then, 15 mature and uniform seeds were selected, one seed per pot. All pots were randomly placed in the incubator under 14 hours of light and 10 hours of darkness, maintaining the above conditions until the end of the incubation period (40 days).

[0054] After incubation, growth conditions were photographed using a digital camera (Canon 750D); pea seedling height (cm) was measured, and roots and buds were cut and plants collected. Collected plant samples were dried at 70.0℃, and dry weight (DW) was measured. The samples were ground into powder, and nicotine and nornicotine concentrations were determined. Soil samples were also collected and divided into two parts. One part was stored at -20.0℃ for nicotine, nornicotine concentrations and soil microbial characteristics were measured; the other part was air-dried to determine its physicochemical properties. The effects of the CDBC and MSBC groups on pea plant growth in nicotine-contaminated soil are shown below. Figure 1 As shown.

[0055] In this application, the method for reducing nicotine pollution of intercropped crops in flue-cured tobacco fields includes the following specific steps:

[0056] 1) Preparation of corn stalk and cow dung biochar: Corn stalk and cow dung were placed in a muffle furnace, and nitrogen was introduced into the vacuum furnace at a rate of 0.1 L / min for 10 min. Then, the dried raw materials were heated to 500℃ at a rate of 10℃ / min and held for 4 h. Pyrolytic char was prepared by slow pyrolysis and labeled as CDBC (cow dung biochar) and MSBC (corn stalk biochar), respectively. The prepared cow dung biochar and corn stalk biochar samples were crushed and passed through a 2 mm sieve for later use.

[0057] 2) Material addition ratios: Apply 50-300 kg / mu of cow dung biochar, 20-100 kg / mu of fly ash, and 10-100 kg / mu of wood vinegar. Specifically, for base fertilizer: Apply 50 kg / mu of superphosphate and 25 kg / mu of 15:15:15 compound fertilizer as base fertilizer in shallow furrows. Combine cow dung biochar and fly ash with the base fertilizer application. Simultaneously, apply 2 kg / mu of chlorpyrifos granules (for controlling cutworms and other underground pests), lightly mixing with a hoe to ensure the fertilizer and pesticide are mixed with the soil. Dilute the wood vinegar 10 times and spray before the first irrigation. Spray the wood vinegar between the plant rows, keeping the nozzle 3-5 cm above the ground, and apply evenly.

[0058] After treating the tobacco field, pea planting is carried out, including the following steps:

[0059] 1) Planting time: Peas are generally planted in an intercropping manner, usually when the tobacco has been harvested and there are still 4 to 5 leaves left on the top.

[0060] 2) Field clearing and soil preparation before sowing: Before sowing, remove weeds from the field and clean up fireworks, rotten tobacco leaves, etc. Keep the ditches clear and the field clean. Prepare the soil surface, with a width of 20-30cm.

[0061] 3) Sowing: On the prepared soil surface, dig a shallow furrow 3-5 cm deep. Apply 50 kg of superphosphate and 25 kg of 15:15:15 compound fertilizer per mu (approximately 0.067 hectares) as base fertilizer. Simultaneously, apply 2 kg of chlorpyrifos granules per mu (to control cutworms and other underground pests). Gently mix the fertilizer and pesticide with the soil using a hoe. Sow 5 kg of seeds per mu, spacing them 5 cm apart in the prepared planting furrow; alternatively, sow 2-3 seeds per hole at a spacing of 10-15 cm, planting in single rows.

[0062] 4) Trellising: When the peas grow to 15-20cm tall, trellise them. First, completely remove the buds from the tobacco plants, leaving the tobacco stalks 1.2m high as supports. First, use coarse yarn to pull a horizontal line 0.8-1m above the soil surface on each tobacco stalk, and tie the line tightly and straight to the tobacco stalk. Then, use fine yarn to wrap around each pea plant and hang it on the horizontal line. Not all branches of each plant need to be hung, but at least one branch should be hung.

[0063] 5) Water and fertilizer management: After the vines have been trained, cultivate the entire soil surface shallowly once, leaving shallow furrows near the roots of the plants. Apply 15 kg of urea per mu (approximately 0.067 hectares) as a seedling fertilizer. Before flowering, apply 20 kg of compound fertilizer and 15 kg of urea per mu as a second fertilizer. During the flowering and pod-setting period, in conjunction with pest and disease control, foliar spray 0.5% potassium dihydrogen phosphate 1-2 times, and apply 20 kg of 15:15:15 compound fertilizer per mu as a fertilizer to promote rapid growth of flowers and pods and increase the yield of tender pods.

[0064] In this application, compared with plant-derived biochar, biochar produced from animal manure has more nutrients, a higher pH value, and a higher specific surface area. When combined with fly ash, it should have a higher adsorption and / or degradation capacity in reducing the absorption and accumulation of nicotine and nornicotine in the soil by plants and promoting their dissipation in the soil.

[0065] In this application, the application rate of cow manure biochar is 50–300 kg / mu, fly ash is 20–100 kg / mu, and wood vinegar is 10–100 kg / mu. Insufficient application has a weak effect on improving tobacco field soil, and its improvement on the physicochemical properties of the soil, nicotine pollution from intercropped crops, the structure of the soil rhizosphere microbial community, and the abundance and diversity of bacterial and fungal communities is not significant. Excessive application can cause soil pH to rise and activate microorganisms related to organic nitrogen mineralization in the soil, thereby increasing ammonia volatilization emissions. Furthermore, adding biochar to low-fertility soils will increase the soil C / N ratio, intensifying competition for nutrients between soil microorganisms and plants, and adversely affecting plant growth.

[0066] In this proposed method, biochar and fly ash are applied in combination with base fertilizer, while chlorpyrifos granules are used and diluted wood vinegar is sprayed before irrigation, followed by topdressing before flowering. Biochar, combined with fly ash and wood vinegar, can serve as a fertilizer supplement, improving soil conditions and promoting plant growth. Simultaneously, it can influence the dissipation of tobacco alkaloids in the soil by soil microorganisms, thereby improving nicotine pollution in nicotine-rich soils and reducing nicotine accumulation in intercropped crops in flue-cured tobacco fields.

[0067] In this application, peas are planted in an intercropping manner, sown when the tobacco has 4-5 leaves remaining at the top. If sown too early, the peas will encounter low temperatures during the flowering period, which is not conducive to normal pollination and will not increase pea yield. If sown too late, the harvest will coincide with the heavy frost period, and the pods will be severely damaged by frost, resulting in reduced yield or even complete crop failure. It is also not conducive to the transplanting of tobacco the following year.

[0068] In this application, pre-sowing field clearing ensures unobstructed drainage and a clean field. Soil preparation is necessary because the raised beds are relatively high, requiring leveling to a width of 20-30cm to facilitate sowing and later-stage management. However, the soil must not block the drainage ditches to prevent waterlogging and seedling death after sowing; maintaining unobstructed drainage is crucial. Furthermore, vine training aims to guide the pea vines onto trellises, preventing them from creeping on the ground and reducing disease incidence. After training, the entire soil surface should be lightly tilled once, leaving shallow furrows near the plant roots for easy watering and fertilization. During the growing season, peas are susceptible to both drought and waterlogging; when the soil is dry, watering or shallow irrigation should be done to keep the soil moist.

[0069] In this proposed method, compared to conventional planting, the application of cow manure biochar in combination with fly ash and wood vinegar can improve the physical and chemical properties of the soil, enhance the soil's fertility, improve the structure of the soil's microbial community, and affect the dissipation of tobacco alkaloids in the soil by soil microorganisms. This can improve the nicotine pollution in nicotine-alkalized soils and reduce the accumulation of nicotine in intercropped crops in flue-cured tobacco fields.

[0070] Test Example 1

[0071] The researchers investigated the effects of cow dung biochar and corn stalk biochar treatments on the concentrations of nicotine and nornicotine in pea roots and stems, the translation factor (TF) of nicotine and nornicotine in pea roots and aboveground parts, the dry weight of pea roots and aboveground parts, and the total uptake of nicotine and nornicotine in pea stems and roots. The results are as follows: Figures 2-3 As shown.

[0072] The results showed that, compared with the control (CK), as the amount of biochar added to the soil increased from 1.5% to 3%, both the cow manure biochar group and the corn straw biochar group significantly reduced the nicotine concentration in the aboveground parts of pea plants, decreasing by 73.3–79.1% and 46.2–71.6%, respectively. The nicotine content in the roots decreased by 60.1–85.1% and 80.7–86.6%, respectively. Figure 2 a). Correspondingly, after treatment with cow dung biochar and corn straw biochar, the total bioaccumulation of nicotine in the aboveground parts of pea plants decreased by 48.6%–52.3% and 32.4–40.7%, respectively, and in the roots by 16.6–71.1% and 61.1–69.8%, respectively. Figure 3 a). These results indicate that the cow dung biochar group was more effective than the corn stalk biochar group in reducing nicotine absorption and bioaccumulation in peas.

[0073] Compared with the control (CK), as the amount of biochar added to the soil increased from 1.5% to 3%, both the cow manure biochar group and the corn straw biochar group significantly reduced the nicotine concentration in the aboveground parts of pea plants by 73.6–89.1% and 42.7–70.8%, and the nicotine concentration in the roots by 69.2–85.0% and 81.7–85.6%, respectively. Figure 2 (b) Furthermore, after treatment with cow dung biochar and corn straw biochar, the total mass of nornicotinic biomass accumulated in the aboveground parts of pea plants decreased by 49.5–74.7% and 28.4–38.8%, respectively, while the total mass of nornicotinic biomass accumulated in the roots decreased by 22.1–70.0% and 63.0–67.2%, respectively. Figure 3 (b) Both cow dung biochar and corn stalk biochar reduced the absorption and accumulation of tobacco alkaloids in pea plants, with the cow dung biochar treatment being more efficient in reducing the absorption and accumulation of tobacco alkaloids.

[0074] The cow manure biochar group had no significant effect on the TF values ​​of nicotine and nornicotine, while the TF values ​​of nicotine and nornicotine were 1.7–2.2 times and 2.1–3.2 times that of the control treatment, respectively, when 1.5% and 3.0% corn straw biochar were added. This indicates that the cow manure biochar group was more effective than the corn straw biochar group in reducing the translocation of nicotine and nornicotine to pea seedlings. Figure 2 c).

[0075] Treatments with 1.5% CDBC, 3.0% CDBC, 1.5% MSBC, and 3.0% MSBC increased the whole-plant dry weight of peas by 96.8%, 131.5%, 26.6%, and 110.4%, respectively. Figure 2 d).

[0076] Based on the above results, it can be seen that biochar treatment can significantly delay the bioaccumulation of tobacco alkaloids in pea sprouts (edible organs), and the treatment efficiency of the cow dung biochar group is higher, which will be more helpful in preventing the potential harm of tobacco alkaloids to human health.

[0077] Test Example 2

[0078] Researchers investigated the effects of cow manure biochar and corn straw biochar treatments on the concentrations of nicotine and nornicotine in the soil where peas were grown. The results are as follows: Figure 4 As shown.

[0079] The results showed that 1.5% MSBC had little effect on nicotine concentration, while 3.0% CDBC and 3.0% MSBC significantly increased the concentrations of nicotine and nornicotine in the rhizosphere soil. Compared with the control (CK), the addition of 1.5–3.0% cow manure biochar increased the concentrations of nicotine and nornicotine by 32.0–96.6% and 58.4–175.3%, respectively; the treatment with 1.5–3.0% corn straw biochar increased the concentrations of nicotine and nornicotine by 1.0–26.9% and 70.9–128.5%, respectively. Figure 4 a).

[0080] It is evident that the cow dung biochar and corn straw biochar treatments have great potential to increase the accumulation of tobacco alkaloids in the rhizosphere soil. Moreover, the cow dung biochar treatment is more efficient than the corn straw biochar treatment, and it increases the concentrations of nicotine and nornicotine in the rhizosphere soil more effectively than the corn straw biochar treatment.

[0081] Test Example 3

[0082] Researchers measured the aboveground enrichment factor (SCF) of nicotine and nornicotine in peas and the concentrations of nicotine and nornicotine in the pore water of nicotine-contaminated soil using cow dung biochar and corn straw biochar treatments. The results are as follows: Figure 4 As shown.

[0083] The results showed that the aboveground enrichment factor (SCF) of the cow dung biochar group and the corn straw biochar group was significantly reduced by 79.8–89.5% and 48.3–78.5%, respectively, and the SCF values ​​of nicotine reduction were significantly reduced by 83.6–96.1% and 66.1–87.4%, respectively. Figure 4 b) indicates that biochar treatment can reduce the transport capacity of peas for two tobacco alkaloids from the soil to the stem, and the effect of cow manure biochar is stronger.

[0084] In addition, the researchers also measured the in-situ pore water concentrations of nicotine and nornicotine in the soil treated with cow dung biochar and corn straw biochar. Figure 4 c). The results showed that both the cow dung biochar group and the corn straw biochar group could significantly reduce the concentrations of nicotine and nornicotine in the in-situ pore water, and the reduction efficiency of the cow dung biochar group was higher than that of the corn straw biochar group.

[0085] In summary, the results show that the cow manure biochar group was more effective than the corn straw biochar group in reducing the concentrations of nicotine and nornicotine in in-situ soil pore water.

[0086] Test Example 4

[0087] The researchers investigated the effects of cow dung biochar and corn straw biochar on the decomposition of nicotine in sterilized and unsterilized soils. The results are shown in Table 1 below.

[0088] Table 1. Effects of cow dung biochar and corn straw biochar on nicotine decomposition in sterilized and unsterilized soils.

[0089] deal with <![CDATA[K(day -1 )]]> <![CDATA[R 2 ]]> <![CDATA[T 1 / 2 (day)]]> Sterile soil CK 0.151 0.929 4.59 1.5% CDBC 0.217 0.945 3.19 3% CDBC 0.299 0.962 2.32 1.5% MSBC 0.201 0.977 3.45 3% MSBC 0.241 0.979 2.88 Unsterilized soil CK 0.916 0.857 0.76 1.5% CDBC 0.892 0.972 0.78 3% CDBC 0.861 0.887 0.81 1.5% MSBC 0.879 0.979 0.79 3% MSBC 0.873 0.957 0.79

[0090] The results showed that biochar treatment reduced the half-life of nicotine in sterilized soil. Specifically, corn straw biochar reduced the half-life of nicotine by 1.14–1.71 days, while cow dung biochar reduced it by 1.40–2.27 days. These results indicate that the half-life of nicotine in sterilized soil was reduced, suggesting that abiotic degradation promoted the dissipation of nicotine, and that the cow dung biochar group was more effective.

[0091] The results showed that biochar treatment increased the half-life of nicotine in unsterilized soil. Specifically, corn straw biochar increased the half-life of nicotine by 0.03 days, while cow dung biochar increased it by 0.02–0.05 days. This increase in the half-life of nicotine in unsterilized soil indicates that both cow dung and corn straw biochar groups prolonged the duration of nicotine presence in unsterilized soil. This may be due to the increased adsorption of nicotine by biochar combined with fly ash, reducing the in-situ pore water concentration of nicotine in the soil, and the reduction of soil microbial bioavailability of nicotine by biochar combined with wood vinegar. These results are consistent with the changes in the nicotine degradation rate constant (k) in sterilized and unsterilized soils.

[0092] Test Example 5

[0093] Researchers used single-concentration point experiments to determine the adsorption capacity of nicotine in cow dung biochar and corn straw biochar groups. The results are as follows: Figure 5 As shown.

[0094] Compared with the control (CK), both the cow manure biochar and corn straw biochar treatments showed significantly increased nicotine adsorption, and the soil Qt value increased with increasing biochar addition (Table 2). Specifically, compared with the CK control, when the biochar addition in the soil increased from 1.5% to 3.0%, the adsorption of nicotine by the cow manure biochar group increased by 6.5–8.1%, and that by the corn straw biochar group by 3.2–4.3%. The adsorption capacity of nicotine was 3% CDBC > 1.5% and MSBC > MSBC 1.5%. Therefore, the cow manure biochar treatment showed a stronger adsorption capacity for nicotine than the corn straw biochar treatment.

[0095] Table 2. Soil adsorption capacity for nicotine in treatments with two different biochar groups.

[0096]

[0097] Test Example 6

[0098] The researchers also detected and analyzed the changes in soil bacterial community composition after the addition of cow dung biochar and corn straw biochar from two perspectives: phylum-level community abundance and genus-level community abundance. The results are as follows: Figure 6 As shown.

[0099] Compared to the control (CK), the addition of biochar increased the differences in bacterial communities in nicotine-contaminated soils, and the responses of the cow dung biochar group and the corn straw biochar group to soil microorganisms also differed. For example, both the cow dung biochar and corn straw biochar treatments increased the relative abundance of Proteobacteria, Gemmatimonadota, and Bacteroidota, and decreased the relative abundance of Acidobacteriota, bacteria that were confirmed to be nicotine and / or nicotine-degrading bacteria. Figure 6 a). At the genus level, compared with the CK control, biochar increased the relative abundance of nicotine-degrading bacteria, such as Arthrobacter, Pseudomonas, and Bispora, while decreasing the relative abundance of Sphingomonas. Figure 6 b). In addition, the cow manure biochar treatment significantly increased the relative abundance of Arthrobacter (18.4%, the most dominant genus among the top 20 bacterial genera), while the corn straw biochar treatment significantly decreased its relative abundance (18.4%).

[0100] In this application, researchers studied cow dung biochar and corn stalk biochar and found that biochar, combined with fly ash and wood vinegar, affects the dissipation of tobacco alkaloids in the soil by soil microorganisms, thereby improving nicotine pollution in nicotine-rich soils. This can be used to solve the nicotine pollution problem in existing technologies when intercropping crops in flue-cured tobacco fields. Specifically, this application found that biochar treatment can significantly delay the bioaccumulation of tobacco alkaloids in pea sprouts (edible organs), and the cow dung biochar group (cow dung biochar + fly ash + wood vinegar) has a higher treatment efficiency. It is foreseeable that the solution proposed in this application will help prevent the potential harm of tobacco alkaloids to human health and will help guide how to conduct intercropping of crops in flue-cured tobacco fields.

[0101] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. A method for reducing nicotine pollution from intercropped crops in flue-cured tobacco fields, characterized in that, The method includes the following steps: S1. Preparation of cow dung biochar: Cow dung raw material is placed in a muffle furnace, and nitrogen gas flows into the vacuum furnace at a rate of 0.1-0.3 L / min for 5-15 min. Then the dried raw material is heated to 450-550℃ and held for 3-5 h. Pyrolytic carbon is prepared by slow pyrolysis to obtain cow dung biochar. The prepared cow dung biochar sample is then crushed and passed through a 2 mm sieve for later use. S2. Apply 50±10 kg / mu of superphosphate and 25±5 kg / mu of 15:15:15 compound fertilizer as base fertilizer. Then apply cow manure biochar and fly ash together with the base fertilizer. At the same time, apply 1-3 kg / mu of chlorpyrifos granules. Use a hoe to gently mix the fertilizer and pesticides with the soil. Before the first irrigation, spray wood vinegar solution diluted 8-12 times.

2. The method according to claim 1, characterized in that, The application rate of the cow dung biochar is 50-300 kg / mu, the application rate of the fly ash is 20-100 kg / mu, and the application rate of the wood vinegar is 10-100 kg / mu.

3. A method for intercropping peas in flue-cured tobacco fields, characterized in that, The method includes the following steps: S1. Preparation of cow dung biochar: Cow dung raw material is placed in a muffle furnace, and nitrogen gas flows into the vacuum furnace at a rate of 0.1-0.3 L / min for 5-15 min. Then the dried raw material is heated to 450-550℃ and held for 3-5 h. Pyrolytic carbon is prepared by slow pyrolysis to obtain cow dung biochar. The prepared cow dung biochar sample is then crushed and passed through a 2 mm sieve for later use. S2. Pretreatment of flue-cured tobacco fields: When the flue-cured tobacco has 4-5 leaves left at the top, prepare to sow peas. On the prepared soil surface, dig a shallow furrow 3-5 cm deep. Apply 50±10 kg / mu of superphosphate and 25±5 kg / mu of 15:15:15 compound fertilizer as base fertilizer in the furrow. Then apply cow manure biochar and fly ash together with the base fertilizer. At the same time, apply an additional 1-3 kg / mu of chlorpyrifos granules. Use a hoe to gently mix the fertilizer and pesticides with the soil. Before the first irrigation, spray with wood vinegar solution diluted 8-12 times. S3. Pea planting: Sow 4-6 kg of pea seeds per mu, with a plant spacing of 4-6 cm in the prepared planting furrows; or, sow 2-3 seeds per hole with a hole spacing of 10-15 cm, and plant in single rows. S4. When the peas grow to 15-20cm tall, build a trellis to guide the vines. First, remove the buds on the tobacco plants and leave the tobacco stalks 1-1.5m high as supports. Then, use yarn to pull a horizontal line 0.8-1m above the soil surface on each tobacco stalk. Tie the horizontal line to the tobacco stalk and use yarn to wrap and hang each pea plant on the horizontal line. S5. After the vines are trained, shallowly cultivate the soil surface and leave shallow furrows near the roots of the plants. Apply a seedling fertilizer once with 15±5 kg / mu of urea diluted in water. Before the peas flower, apply 20±5 kg / mu of 15:15:15 compound fertilizer and 15±5 kg / mu of urea once more. During the flowering and pod-setting period of the peas, spray the leaves with 0.5% potassium dihydrogen phosphate 1-2 times and apply 20±5 kg / mu of 15:15:15 compound fertilizer diluted in water.

4. The planting method for intercropping peas in flue-cured tobacco fields according to claim 3, characterized in that, The application rate of cow dung biochar is 50-300 kg / mu, the application rate of fly ash is 20-100 kg / mu, and the application rate of wood vinegar is 10-100 kg / mu.

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